The determination of cellular phenotypes is an essential aspect of development; this process is particularly complex within structures comprised of highly ordered cellular patterns such as the sensory epithelium of the mammalian cochlea. Within the organ of Corti, a single row of inner hair cells and three rows of outer hair cells extend along the basal-to-apical axis of the cochlea. Every sensory hair cell is separated from the next by an intervening non-sensory supporting cell resulting in an invariant mosaic of several varying cell types. The importance of the formation of this structure is illustrated by the significant auditory deficits observed in animals with cochlear patterning defects (Barald and Kelley, 2004). Considering the importance of the basic biological function of the auditory system as well as its value as a model organ system, the long term goal of our research is to uncover and elucidate the molecular signaling pathways responsible for the development of the mammalian cochlear sensorineural array. Among the transduction pathways that regulate cellular determination are the Wnt signaling cascades. The effects of Wnt signaling are varied, dependent on tissue and cell type, and act within a developmental context. Wnt signaling has been shown to activate three main downstream pathways: the well-characterized Wnt/-catenin pathway also known as the canonical pathway, and the two less-characterized noncanonical pathways: the Jun-N-terminal kinase pathway, also known as the planar cell polarity pathway, and the Wnt/Calcium/PKC pathway (Wodarz and Nusse, 1998; Huelsken and Birchmeier, 2001; Kuhl, 2004; Nusse, 2005). Based on our preliminary results, we will test the hypothesis that the Wnt/Calcium/PKC signaling cascade functions in determining cell fate in the developing cochlea. Our data indicate that: 1) Wnt4 inhibits hair cell formation, 2) Wnt4 signals through the Wnt/Calcium/PKC pathway, 3) PKC regulates cell fate in the cochlear duct, and 4) the activity of Atoh1 can be regulated through PKC phosphorylation. From this preliminary data we have devised a working model which suggests that a Wnt4 gradient located at the medial edge of the cochlear duct plays a role in regulating the boundary between sensory and non-sensory regions through inhibition of hair cell formation. Wnt4 signals through PKC; the kinase, in turn, phosphorylates Atoh1 and inhibits its activity. We further hypothesize that Wnt4 activity is antagonized by a sFRP2 (a Wnt inhibitor) gradient originating from the lateral edge of the cochlea duct, and that this antagonism is integrated to specify the medial hair cell boundary. The following specific research aims will be used to test these hypotheses: Specific Aim 1: We will investigate if the Wnt/Calcium gene Wnt4 is involved in determining cell fate in the cochlea. We will determine the spatiotemporal expression of Wnt4 in the developing cochlea and investigate its gradient and activity; we will analyze the cellular and morphological defects of Wnt4 mutant cochleae. Our preliminary observations show the formation of extra inner hair cells in Wnt4 mutants. We will also perform gain- and loss-of-function experiments in vitro to further elucidate the role of Wnt4. Specific Aim 2: We will characterize the interaction between Wnt4 and the Wnt inhibitor sFRP2 in the cochlea. We will elucidate the Wnt4 signaling cascade using in vitro and biochemical experiments. Our preliminary data indicate that Wnt4 signals through the Wnt/Calcium/PKC pathway in the embryonic cochlea and that inhibition of this pathway results in the development of extra inner hair cells phenocopying the Wnt4 mutant. We will investigate the downstream components and modulators of this signaling pathway. Specific Aim 3: We will determine if PKC, directly activated by Wnt4, has the ability to phosphorylate Atoh1 and will determine the effect of this phosphorylation on Atoh1 activity. PUBLIC HEALTH RELEVANCE: Deafness is one of the most common birth defects in humans and many forms are due to abnormalities in the developmental processes that are necessary for the formation of the cochlea. The goal of this project is to determine the molecular mechanism and role of the important Wnt/Calcium/PKC pathway in auditory hair cell formation. These studies will provide novel insights into cochlear development that might facilitate the advancement of strategies for the regeneration of mechanosensory hair cells that could potentially result in the improvement of hearing.